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Vol. 14, Issue 6, 2470-2481, June 2003
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3Cx46 and 8Cx50 Interact with Zonula Occludens Protein-1 (ZO-1)
#
* Department of Cell Biology, The Scripps Research Institute, La Jolla,
California 92037, USA;
Department of Ophthalmology and Visual Sciences, Washington University School
of Medicine, St. Louis, Missouri 63110, USA;
Division of Cellular Biochemistry, The Netherlands Cancer Institute,
Amsterdam, The Netherlands;
Institut Jacques Monod, CNRS-Universités Paris 6-Paris 7, Paris,
France; and
|| Department of Ophthalmology and Visual Sciences, University of Illinois at
Chicago, Chicago, Illinois 60612, USA
Submitted October 8, 2002;
Revised January 20, 2003;
Accepted February 26, 2003
Monitoring Editor: Daniel Goodenough
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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1Cx43 has previously been shown to bind to the PDZ
domain–containing protein ZO-1. The similarity of the carboxyl termini
of this connexin and the lens fiber connexins 3Cx46 and 8Cx50
suggested that these connexins may also interact with ZO-1. ZO-1 was shown to
be highly expressed in mouse lenses. Colocalization of ZO-1 with 3Cx46
and 8Cx50 connexins in fiber cells was demonstrated by
immunofluorescence and by fracture-labeling electron microscopy but showed
regional variations throughout the lens. ZO-1 was found to coimmunoprecipitate
with 3Cx46 and 8Cx50, and pull-down experiments showed that the
second PDZ domain of ZO-1 was involved in this interaction. Transiently
expressed 3Cx46 and 8Cx50 connexins lacking the COOH-terminal
residues did not bind to the second PDZ domain but still formed structures
resembling gap junctions by immunofluorescence. These results indicate that
ZO-1 interacts with lens fiber connexins 3Cx46 and 8Cx50 in a
manner similar to that previously described for 1Cx43. The spatial
variation in the interaction of ZO-1 with lens gap junctions is intriguing and
is suggestive of multiple dynamic roles for this association. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). A gap junction channel consists of two interacting
hemichannels (connexons), which contain six connexin subunits each. Each
connexin is a polypeptide that traverses the cell membrane four times, with
both the NH2- and COOH-termini located in the cytoplasm
(Milks et al., 1988;
Yeager and Gilula, 1992).
Connexons may consist of only one connexin isoform (homomeric) or of multiple
isoforms (heteromeric). In the ocular lens, cells in the interior are
dependent on gap junctional communication to maintain the ionic and water
balance of the intercellular milieu, and the transparency and optical
properties of the lens (Mathias et
al., 1997). The anterior epithelial monolayer of the lens
contains gap junctions composed primarily of 1Cx43 connexin, whereas
3Cx46 and 8Cx50 connexins are coexpressed during the process of
terminal differentiation and elongation of the epithelium into fiber cells.
The importance of a functional gap junction network in the lens is
demonstrated by targeted deletion of 3Cx46 connexin, which results in
nuclear cataracts and Ca2+-activated proteolysis
(Gong et al., 1997;
Baruch et al., 2001),
whereas 8Cx50-knockout mice show microphthalmia and develop a
pulverulent type of cataract (White et
al., 1998).
In lens fiber cells, 3Cx46 and 8Cx50 connexins are found in
the same junctional plaque (Paul et
al., 1991; Dunia et
al., 1998) and have been reported to form heteromeric
connexons (Jiang and Goodenough,
1996). In the lens cortex, these connexins are localized primarily
to the broad sides of fiber cells
(Gruijters et al.,
1987; Tenbroek et
al., 1992), whereas in the nuclear region, the COOH-termini
of 3Cx46 and 8Cx50 connexins are proteolytically cleaved, and
the packing arrangement of the junctional plaques is modified. This ordered
distribution of gap junctions is thought to be important for maintaining lens
homeostasis because of involvement in a proposed internal microcirculatory
system (Mathias et al.,
1997). The details of how the organization and processing of lens
gap junctions are achieved are unclear.
In a multitude of cellular systems containing specialized membrane domains,
certain membrane channels and receptors have been demonstrated to interact
with proteins containing PDZ (PSD-95, discs large, ZO-1) domains (e.g., Shaker
voltage-gated K+ channels [Kim
et al., 1995] and 
2-adrenergic receptors
[Hall et al.,
1998a]). In some cases, these interactions are needed to direct
the membrane proteins to the appropriate membrane subdomain
(Muth et al., 1998;
Moyer et al., 2000).
Other roles for PDZ domain–containing proteins include coupling channels
and transmembrane proteins to downstream signaling and cytoskeletal elements
and their involvement in insertion, endocytosis, and recycling of proteins
(Fanning et al., 1999). Zona occludens protein-1 (ZO-1) is a member
of the MAGUK (membrane-associated guanylate kinase) family and contains three
PDZ domains, an Src-homology-3 (SH3) domain, and an inactive guanylate kinase
(GUK) domain. The interaction of ZO-1 with the tight junction components
occludin and claudins and with cadherins has been demonstrated previously
(Itoh et al., 1993).
ZO-1 also interacts with 1Cx43 connexin
(Toyofuku et al.,
1998) via binding of the second PDZ domain to the most
COOH-terminal residues of 1Cx43
(Giepmans and Moolenaar, 1998;
Giepmans et al.,
2001). Recently, 7Cx45 was also shown to interact with
ZO-1, although it is not clear what domains are involved in this interaction
(Kausalya et al.,
2001; Laing et al.,
2001).
Because of the sequence similarity between the COOH-termini of
1Cx43, 3Cx46, and 8Cx50, the expression of ZO-1 and its
interaction with 3Cx46 and 8Cx50 connexins was examined in the
lens.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Antibodies
A rabbit pAb was raised against a synthetic peptide from the cytoplasmic
loop of mouse 8Cx50 as described previously
(White et al., 1992)
and affinity-purified. A rabbit pAb was raised against a synthetic peptide
from the cytoplasmic loop of mouse 3Cx46 (RRDNPQHGRGREPMC) and
affinity-purified. This antibody has been used in previous studies
(Gong et al., 1997;
Dunia et al., 1998).
An anti–ZO-1 pAb was obtained commercially (Zymed Laboratories, South
San Francisco, CA). A rat anti–ZO-1 monoclonal antibody (mAb), R26.4C
(Stevenson et al.,
1986; Anderson et al.,
1988), was obtained from the Developmental Studies Hybridoma Bank
developed under the auspices of the National Institute of Child Health and
Human Development and maintained by The University of Iowa, Department of
Biological Sciences, Iowa City, IA.
Immunoblot Analyses
Lens material was homogenized in IP buffer (50 mM Tris, 150 mM NaCl, 2 mM
EDTA, 1% NP-40, 0.25% deoxycholate, pH 8.0), sonicated, and clarified by
centrifugation. Samples were separated on 7% or 10% SDS-PAGE gels on a Hoefer
vertical gel apparatus, followed by transfer to Protran 0.2-µm pore size
nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Membranes were
stained with 0.2% Ponceau S in 1% acetic acid, blocked with 5% skimmed milk
powder in TBST, and incubated with primary antibodies. These were detected by
chemiluminescence (SuperSignal West Pico, Pierce, Rockford, IL) using goat
anti-rabbit horseradish peroxidase–conjugated secondary antibodies
(Bio-Rad Laboratories, Hercules, CA), followed by exposure to Biomax ML film
(Eastman Kodak).
Immunostaining and Confocal Laser Scanning Microscopy
Lenses from C57BL/6 mice were fixed in 4% paraformaldahyde for 40 min,
sectioned to a thickness of 150 µm with a Vibratome (model 3000, TPI, St.
Louis, MO), and refixed in 4% paraformaldahyde for 30 min. For most of the
colocalization stainings, rabbit pAbs were used to detect 8Cx50 and
3Cx46, as well as ZO-1, by use of a procedure that discriminated
between the various antibodies. Briefly, sections were blocked with 5% goat
serum in PBS, followed by application of the connexin antibody. The bound
antibody was detected with goat anti-rabbit Fab fragments conjugated with
rhodamine (Jackson ImmunoResearch Laboratories, West Grove, PA). Bound pAbs
were then blocked with unconjugated goat anti-rabbit Fab fragments, after
application of anti-ZO-1 pAbs. The bound ZO-1 antibody was then detected with
Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR).
Controls included incubation with either of the primary antibodies alone,
followed by incubation with Alexa 488-conjugated secondary antibody, blocking
with Fab fragments, and incubation with rhodamine-conjugated secondary
antibody. In all cases, very little staining was observed from the
rhodamine-conjugated secondary antibody, indicating that the blocking was
efficient and that the labeling of individual primary antibodies was specific.
Furthermore, these results were verified by substituting the rabbit pAb
against ZO-1 with rat mAb R24.6C and detecting the primary antibodies with
species-specific secondary antibodies. In this case, R24.6C was detected by
use of goat anti-rat FITC (Southern Biotechnology Associates, Birmingham, AL).
Stained preparations were imaged with a confocal microscope (LSM410, Carl
Zeiss, Thornwood, NY) equipped with an argon/krypton laser.
Cell Lines. Cells were grown on poly-L-lysine–treated glass coverslips, washed in PBS, and fixed in -20°C methanol for 6 min. Coverslips were blocked with 5% goat serum in PBS (blocking buffer), followed by incubations with primary antibodies overnight in blocking buffer at 4°C. pAbs were used at 1–5 µg/ml. After washing with PBS, coverslips were incubated with fluorochrome-labeled secondary antibodies (Southern Biotech Associates, Birmingham, AL), diluted 1:100, together with 50 nM To-Pro3 (Molecular Probes, Eugene, OR), in blocking buffer. Washed slides were mounted with Fluoromount-G (Sigma, St. Louis, MO), and images were collected with a Zeiss Axiovert confocal microscope.
Electron Microscopy Freeze-Fracture Immunolabeling
Lenses for freeze-fracture immunolabeling were dissected immediately after
the animals were killed. The cortical lens region was separated, and small
pieces were placed on flat gold specimen holders (Balzers, Liechtenstein),
frozen by quick immersion in liquid propane (Balzers), and finally stored in
liquid nitrogen until replicated. Freeze-fracture was performed at -140°C
in a freeze-fracture apparatus (model 301 or 400; Balzers). After fracture,
the specimens were shadowed by platinum/carbon evaporation from an electron
gun. The replicas were detached from the tissue by immersion in PBS, treated
with 2% SDS, and processed for immunolabeling according to a technique
described elsewhere (Dunia et
al., 2001). Replicas were examined with a Philips CM12 or
Tecnai 12 electron microscope operating at 80 kV.
Immunoprecipitation
Lenses from adult wild-type C57BL/6, 8Cx50-/-, and 3Cx46-/-
mice were homogenized in IP buffer, as described above. This buffer has been
optimized to maximize the binding of 1Cx43 to ZO-1 while minimizing
nonspecific interactions (Giepmans and
Moolenaar, 1998). The clarified homogenates were incubated with
either mAb R26.4C (anti–ZO-1) or normal rat serum bound to protein-G
agarose. The agarose beads were washed extensively in IP buffer and eluted
with SDS-PAGE sample buffer, and precipitated proteins were analyzed by
immunoblotting.
Constructs
The generation of ZO-1 PDZ–GST fusion protein constructs was
described previously (Nielsen et
al., 2002).
Oligonucleotide primers A3FOR
(5'-ATGGGATCCGCAATGGGCGACTGGAGCTTCC-3') and A3REV
(5'-ATGGAATTCCTAGATGGCCAAGTCACCTGGTCTGGC-3') were used to amplify
the coding region of 3Cx46 from mouse genomic DNA by PCR. Similarly,
the coding region of 3Cx46 lacking the most COOH-terminal isoleucine
residue (
I) was amplified with primers A3FOR and A3dIREV
(5'-ATGGAATTCCTAGGCCAAGTCACCTGGTCTGGC-3'). The coding region of
8Cx50 was amplified with primers A8FOR
(5'-ATGGGATCCGCAATGGGCGACTGGAGTTTCC-3') and A8REV
(5'-ATGGAATTCTCATATGGTGAGATCATCTGACCTGGC-3'), and the
8Cx50I construct was generated with primers A8FOR and A8dIREV
(5'-ATGGAATTCTCAGGTGAGATCATCTGACCTGGC-3'). The PCR products were
digested with BamHI and EcoRI and cloned into pcDNA3
(Invitrogen, San Diego, CA). This expression vector contains a CMV promotor
and a SV40 polyadenylation signal. After sequence verification, the constructs
were transiently expressed in HEK293 cells.
Pull-down Experiments
Lenses from 2- to 4-wk-old wild-type C57BL/6 and 3Cx46 and
8Cx50 knockout mice were homogenized in IP buffer and clarified. GST
fusion proteins containing the PDZ domains of ZO-1 were induced by standard
procedures and bound to glutathione-agarose. After washing, agarose beads were
incubated with equal amounts of lens homogenate. Agarose beads were then
washed extensively, and fusion proteins, together with specifically bound
proteins, were released from the beads with SDS-PAGE sample buffer. The
samples were analyzed by immunoblotting. Membranes were stained with Ponceau S
to verify that equal amounts of the PDZ1-, 2-, and 3-GST fusion proteins had
been incubated with lens homogenates.
|
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
|
ZO-1 protein levels were then analyzed by immunoblot of lysates from several mouse tissues (Figure 1B) and were found to correlate well with the respective ZO-1 RNA levels in Figure 1A, confirming that significant amounts of ZO-1 were expressed in lens (Figure 1B).
In these studies, whole lenses were analyzed. These contain at least three regions, epithelium, cortical fibers, and nuclear fibers, with cells at different stages of differentiation. To determine the proportion of ZO-1 in each of these regions, mouse lenses were microdissected to enrich for the respective layers, and the fractions were analyzed by immunoblot. ZO-1 was found in all regions of the mouse lens, although more abundantly in the epithelial and differentiating, cortical fiber cell layers (Figure 1C, lanes 1–2) than in the mature, nuclear fiber cells (Figure 1B, lane 3). Furthermore, immunoblot analysis of whole lenses from 1-, 2-, 3-, and 16-wk-old mice revealed that ZO-1 levels decreased drastically 3 wks after birth (Figure 1D).
Localization of ZO-1 and Connexins in the Lens
Mouse lenses were sectioned in the equatorial plane and examined by
immunofluorescence-scanning confocal microscopy with two different antibodies
against ZO-1. These two antibodies gave similar staining patterns. Distinct
zones with different ZO-1 staining patterns were discernible in the lens and
are labeled by numbers (1–4) in
Figure 2. A description of the
observed results in the different zones is given below.
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Zone 1. At the epithelium–fiber interface, an intense punctate staining at the apical side of the epithelial cells was observed from the proliferative region to the equator. At high magnification, a punctate staining was also observed at cell–cell contacts between epithelial cells. Similar staining was also observed at cell–cell contacts between the posterior tips of elongating fibers. It is noteworthy that such punctuate staining of ZO-1 at the fiber cell tips was observed only at the posterior part of the lens. At the corresponding domain of the epithelial cell membrane, staining for ZO-1 was not observed (our unpublished results).
Zone 2. In outer cortical fibers cells, 10–150 µm from the surface, a punctate membrane staining with spots predominantly at the narrow faces of fiber cell hexagons was observed. For a few outer cell layers, small, scattered punctuate spots of ZO-1 were seen at the broad face of fiber cells, but these disappeared by 30–40 µm and deeper (our unpublished results).
Zone 3. At 175–300 µm from the surface (midcortex), ZO-1 appeared to translocate from the narrow side of fiber cells, to localize predominantly to the broad face of fiber cells.
Zone 4. At 325–425 µm from the surface (deep cortex), ZO-1 appeared to be more evenly distributed on both narrow and broad sides of fiber cells, although these are more irregular at this location.
In the nuclear region, a diffuse staining of the plasma membrane was observed. At high magnification, however, it became apparent that ZO-1 staining was not uniform across the fiber cell membrane, showing limited areas of decreased staining. Because of the extensive proteolysis and exposure of cryptic epitopes in the nucleus, it is not clear whether the staining observed in the nucleus represents intact ZO-1, fragments of ZO-1, or cross-reactivity of the antibodies (our unpublished results).
The colocalization of ZO-1 with 3Cx46 and 8Cx50 connexins in
mouse lens sections was then examined
(Figure 3). In the outer cortex
(corresponding to zone 2 in Figure
2, 10–150 µm from the surface), ZO-1 and 3Cx46
connexins are located primarily on different faces of hexagonal fiber cells:
ZO-1 was observed on the narrow faces, whereas 3Cx46 connexins
localized to the broad faces of fiber cells
(Figure 3, A–C; detail in
Figure 3, D–F). In the
midcortex (corresponding to Zone 3 in
Figure 2, 175–300 µm
from the surface) and continuing into the deep cortex (corresponding to Zone 4
in Figure 2, 325–425
µm from the surface), ZO-1 was observed to colocalize extensively with
3Cx46 on the broad faces of fiber cells
(Figure 3, G–I).
Furthermore, at the lens periphery, some colocalization at the
fiber–epithelium interface and the lateral membrane was observed (our
unpublished results).
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Similarly to 3Cx46, 8Cx50 connexin was observed to colocalize
extensively with ZO-1 in both mid and deep cortex but rarely in the outer
cortex, where 8Cx50 was also located primarily at the broad faces of
fiber cell hexagons (Figure 3,
J–L).
Topographic distribution of ZO-1 and 3Cx46 and 8Cx50
Connexins as Revealed by Fracture-Labeling
Electron microscopy analysis of fracture-labeling (FL) of lenses performed
with either anti-3Cx46 or anti-8Cx50 connexin antibodies, or
both, indicated that these connexins are the major constituents of the nascent
junctional domains (linear strands or small packed arrays of 9-nm junctional
intramembranous particles). On the protoplasmic fracture face (PF), the
fracture exposed large aggregates of 9-nm intramembranous particles and the
corresponding pits on the exoplasmic fracture face (EF), which are a
characteristic of gap junctions. The fiber connexins appeared codistributed on
the same junctional plaque. Double-gold immunolabeling with anti-3Cx46
or anti-8Cx50 and anti-ZO-1 antibodies demonstrated that both ZO-1 and
connexins could be detected in the same junctional plaque
(Figure 4A). At more advanced
stages of junctional assembly, double-gold immunolabeling with anti–ZO-1
antibodies and anti-3Cx46 (Figure
4B) or anti-8Cx50
(Figure 4C) indicated that ZO-1
is randomly distributed within the junctional plaque. In a few junctional
domains, the gold-labeled ZO-1 appeared preferentially packed at the periphery
of the junctional plaque. However, ZO-1 does not form a crown of labeled
particles along the edge between EF and PF as has been described previously
for MP26 (Dunia et al.,
1998).
|
FL on Lens Fiber Cells from Mice Lacking Either 3Cx46 or
8Cx50 Connexins
Targeted gene ablation of lens connexin produces two different phenotypes:
a nuclear cataract in 3Cx46 (-/-) mice and a microphthalmia associated
with a pulverulent type of cataract in 8Cx50 (-/-) mice. The
topographic distribution of the immunogold-labeled ZO-1 in the lens fiber
cells of each of the connexin knockout mice is comparable to that described
above for wild-type mouse lenses. Thus, double-immunogold labeling using
anti-3Cx46 or anti-8Cx50 and anti-ZO-1 antibodies, respectively,
indicated that ZO-1 remains in close topographic association with the
junctional plaques irrespective of whether they contained
homomeric–homotypic 8Cx50 connexons (3Cx46-/-)
(Figure 4D) or
homomeric–homotypic 3Cx46 connexons (8Cx50 -/-)
(Figure 4E).
Coimmunoprecipitation of ZO-1 and Lens Connexins
To determine whether ZO-1 interacts with 3Cx46 and 8Cx50 in
mouse lenses, immunoprecipitated ZO-1 from mouse lenses was analyzed by
immunoblotting using specific connexin antibodies. These experiments
demonstrated that 3Cx46 was coprecipitated with ZO-1 from total lens
lysates (Figure 5A, lane 3) but
not with an irrelevant antibody (Figure
5A, lane 2). A similar coimmunoprecipitation of 3Cx46 with
ZO-1 was observed with lens lysates prepared from 8Cx50 knockout mice
(Figure 5A, lane 4), suggesting
that this is a result of direct interactions with 3Cx46 connexin and
not of heteromeric gap junctions of 3Cx46 and 8Cx50 connexins
that may exist in the lens. Reprobing of the blot showed that ZO-1 was present
in both immunoprecipitations, as expected
(Figure 5A, bottom). A similar
immunoprecipitation of ZO-1 from wild-type mouse lenses and from 3Cx46
knockout lenses showed that 8Cx50 connexin also coprecipitated with
ZO-1 but not with an irrelevant antibody
(Figure 5B). Ponceau staining
of the blot showed that equal amounts of anti–ZO-1 and irrelevant
antibody had been used for the immunoprecipitation
(Figure 5B, bottom).
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The Second PDZ Domain of ZO-1 Interacts with Lens Connexins
The different PDZ domains of ZO-1 were analyzed for their involvement in
the interaction with 3Cx46 and 8Cx50 connexins. Lysates from
mouse lenses were used in pull-down experiments with the three separate ZO-1
PDZ domains expressed as GST fusion proteins, and the lens connexins bound to
the fusion proteins were detected by immunoblot analysis. Using the second
PDZ-domain of ZO-1, a signal for both 3Cx46
(Figure 6A, lane 3) and
8Cx50 (Figure 6B, lane
3) was observed, suggesting its involvement in the interaction with lens gap
junctions. No interaction between lens connexins and the first or third PDZ
domain was detected (Figure 6, A and
B). Ponceau staining of the blot showed that equal amounts of
PDZ-GST fusion proteins were used in all pull-down experiments
(Figure 6, A and B, bottom). To
determine whether these results were a result of the presence of the other
connexin isoform in a heteromeric connexon, similar pull-down assays were
performed using the second PDZ domain and lens lysates from 3Cx46 and
8Cx50 knockout mice. As expected, 3Cx46 was detected in
8Cx50 knockout lenses (Figure
6C, lane 2) but not 3Cx46 knockout lenses
(Figure 6C, lane 1). Pull-down
experiments with the second PDZ domain and lens lysates from these knockouts
showed that 3Cx46 could bind to the second PDZ domain in the absence of
8Cx50 (Figure 6C, lane
4). Similarly, 8Cx50 was found to bind to the second PDZ domain in the
absence of 3Cx46 (Figure
6D, lane 3), excluding the possibility that the binding was a
result of the presence of 3Cx46 in heteromeric connexons. The presence
of equal amounts of PDZ2-GST fusion protein in the pull-down assays was
demonstrated by Ponceau staining of the blot
(Figure 6, C and D,
bottom).
|
The COOH-Terminal Residues of Lens Connexins Bind to ZO-1
It has been demonstrated previously that the most COOH-terminal residue is
involved in the interaction of 1Cx43 with ZO-1, because deleting this
residue abolishes binding (Giepmans and
Moolenaar, 1998). To determine the involvement of the most
COOH-terminal domains of lens connexins in the interaction with ZO-1,
constructs encoding mouse 3Cx46 and 8Cx50 and mutants of these
lacking the most COOH-terminal isoleucine residues (3Cx46I and
8Cx50I) were generated and transiently overexpressed in HEK293
cells. Immunofluorescence examination of these cells using anti-connexin
antibodies revealed fluorescent spots consistent with the formation of gap
junctions between two adjoining cells at sites of cell–cell contact of
some cell pairs expressing either 3Cx46
(Figure 7A, arrow) or
3Cx46I (Figure
7B, arrow). Similar immunofluorescent staining was detected
between some cell pairs expressing either 8Cx50 or 8Cx50I
(Figure 7, C and D, arrows),
indicating that these mutants retain the ability to traffic and assemble into
what are probably gap junctions in HEK293 cells. Some cell pairs
overexpressing either the wild-type or mutated connexins showed intense
fluorescence spread all over the plasma membrane and accumulation within
cellular compartments, most likely because of high levels of connexin protein
expressed in these cells (Figure
7D).
|
When homogenates of these cells were analyzed by immunoblotting using
connexin antibodies, products were detected for all four constructs but not in
wild-type HEK293 cells, as expected (Figure
8, A and B, bottom). However, analysis of homogenates from cells
transfected with 3Cx46 and 3Cx46I indicated the presence
of several products, most of which migrated slightly faster than lens
3Cx46 in SDS-PAGE (Figure
8A, bottom, lanes 3 and 4, arrow). In contrast, a single connexin
band was observed by SDS-PAGE in homogenates from cells expressing
8Cx50 and 8Cx50I after short exposure. The SDS-PAGE
mobility of this band was similar to the upper, major band of 8Cx50
from lens lysate (Figure 8B,
bottom, lanes 3 and 4). Ponceau staining of the blots showed that equal
amounts of lens, wild-type HEK293, and transfected HEK293 lysates were
analyzed (Figure 8, A and B, top). Homogenates from HEK293 cells overexpressing both the wild-type and
mutant connexins were then used in pull-down assays with the second PDZ domain
of ZO-1 fused to GST. These experiments demonstrated that both wild-type
3Cx46 and wild-type 8Cx50, overexpressed in HEK cells, bound to
the second PDZ-domain as expected (Figure
8, A and B, bottom, lanes 5). In contrast, neither
3Cx46I nor 8Cx50I bound to the second PDZ-domain
(Figure 8, A and B, lane 6).
Ponceau staining of the blot showed that equal amounts of PDZ2 had been used
for the pull-down experiment (Figure 8, A
and B, top, lanes 5 and 6, arrowhead).
|
These results indicated that the most COOH-terminal isoleucine residues in
both connexins were involved in the interaction with ZO-1. Interestingly, for
3Cx46, a shorter exposure of the blot showed that primarily the
slowest-migrating forms of 3Cx46 in SDS-PAGE bound to PDZ2. These
connexin isoforms were thus enriched for in the pull-down assay, because they
could be discerned only in the total cell lysates on longer exposure (our
unpublished results). In contrast, the fastest-migrating forms, which were the
most abundant forms observed in the cell lysate
(Figure 8, bottom, arrow), were
underrepresented in the pull-down. A possible explanation for this could be
that most 3Cx46 is proteolytically cleaved in HEK293 cells, which would
explain the much faster mobility compared with the lens lysate isoform. This
cleavage takes place from the COOH-terminus of 3Cx46, similar to the
proteolytic cleavage observed in mature layers of older lenses. In this case,
the cleaved isoforms would be expected to be underrepresented or absent in the
PDZ2 pull-down because of their lack of PDZ binding domain. Their presence in
the pull-down, however, may be a result of their presence in connexons
containing some proportion of the full-length connexin isoforms.
In contrast to 3Cx46, the amount of the faster-migrating isoform of
8Cx50 in the PDZ2 pull-down was approximately similar to the amount
present in the cell lysate, as seen after longer exposure of the blot (our
unpublished results). One possible explanation for this is that the two
isoforms of 8Cx50 differ only in phosphorylation, which potentially
does not affect binding to PDZ2.
|
DISCUSSION |
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|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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3Cx46 and 8Cx50 via a
molecular interaction involving the second PDZ domain of ZO-1 and the most
COOH-terminal residue of each connexin.
Expression and Topographic Distribution of ZO-1, 3Cx46, and
8Cx50 in the Lens
Because ZO-1 has been found primarily in association with tight junctions,
which are present in the lens only at the epithelium–fiber interface, at
least in some species (Zampighi et
al., 2000), it was surprising that lens contained high levels
of ZO-1 RNA and protein compared with other organs.
In the outer cortex, ZO-1 is found primarily at the narrow faces of fiber
cells, whereas in mid to deep cortex, ZO-1 is translocated to the broad face
of fiber cells. In accordance with this finding, double-immunofluorescence
staining revealed that ZO-1 is colocalized with lens fiber cell connexins
3Cx46 and 8Cx50 to various extents in different regions of the
lens. The colocalization ranged from limited in the outer cortex to more
extensive in the midcortex. The colocalization was observed primarily at
junctional domains on the broad faces of fiber cells and not on the narrow
faces of the fibers, which contain a limited number of gap junctions. These
findings are consistent with the relatively low amount of lens connexins that
coimmunoprecipitated with ZO-1 in our experiments, in which whole lenses were
used. The extent of colocalization between 1Cx43 and ZO-1 in cardiac
myocytes has also been reported to be limited
(Barker et al., 2002),
suggesting that in the lens and heart, a large proportion of the connexin pool
does not interact stably with ZO-1, and vice versa.
It is likely that ZO-1 interacts with other proteins besides connexins in
the lens, as has been demonstrated in other cell and tissue types. Thus, ZO-1
has been reported to interact with, e.g., catenin
(Rajasekaran et al.,
1996), JAM (Bazzoni et
al., 2000), cadherins
(Itoh et al., 1993),
and actin filaments (Itoh et al.,
1997). Interestingly, the high levels of ZO-1 detected in lens
epithelial cells by immunoblot analysis was found by immunofluorescence to
localize primarily to the apical side of these cells, i.e., at the
epithelium–fiber interface. It is a distinct possibility that ZO-1 in
these cells interacts with 1Cx43, but ZO-1 probably also interacts with
adhesion molecules that anchor the epithelial cell layer to the fiber cells.
Furthermore, actin is an integral constituent of the plasma
membrane–cytoskeleton complex of lens fibers. To verify and further
examine the presence of ZO-1 in cortical fiber cells, fiber ghost cell
preparations containing the plasma membrane cytoskeleton
(Benedetti et al.,
1996) were stained for ZO-1. ZO-1 was found to show a patchy
distribution along the fiber cell plasma membrane and forming a row of
variously sized plaques in the membrane profile between two adjoining fiber
cells (our unpublished results). These findings demonstrate that ZO-1 remains
associated with the fiber cell ghost preparation after extraction of the
water-soluble fiber constituents, suggesting that ZO-1 may be part of the
plasma membrane–cytoskeleton complex. ZO-1 is probably not involved in
anchoring gap junctions to the cytoskeleton in the rodent lens, because only
primate and human lenses show association between gap junctions and actin
filament bundles, whereas rodent lenses do not
(Lo et al.,
1994).
Potentially, ZO-1 could coordinate the organization of specialized membrane
domains and/or signaling mechanisms, because other members of the MAGUK family
are implicated in the control and assembly of specialized membrane domains
(Fanning et al.,
1998; Fanning and Anderson,
1999; Baruch and Lim,
2001). In the heart, 1Cx43 connexin is localized primarily
to the intercalated disk in cardiac myocytes, and it is thought that
interaction between 1Cx43 and ZO-1 is needed to localize the connexin
to this specialized membrane domain
(Toyofuku et al.,
1998). A similar localization of gap junctions to unique membrane
domains is also found in, e.g., polarized thyroid epithelial cells
(Guerrier et al.,
1995) and in the lens, in which gap junctions in the cortex
localize primarily to the broad face of lens fiber cells. It is interesting
that we observed only limited colocalization of ZO-1 with 3Cx46 or
8Cx50 in the outer cortex, which is the region of the lens in which gap
junction plaques are organized, whereas more extensive colocalization was
observed in midcortex, in which mature gap junction plaques are present. One
explanation for this could be that the association of ZO-1 with 3Cx46
or 8Cx50 has diverse roles in the different regions of the lens.
Perhaps only limited amounts of ZO-1 are necessary to organize and direct the
gap junctional plaques to their correct membrane localization in the outer
cortex, if ZO-1 is even involved in this process. In contrast, it is striking
that the more extensive association between ZO-1 and 3Cx46 and
8Cx50 deeper in the cortex occurs at a stage of lens fiber development
that precedes the proteolytic cleavage of the COOH-terminal of both
3Cx46 and 8Cx50. One intriguing possibility is that ZO-1 binding
to lens connexins may be involved in coordinating or targeting the activities
of proteolytic enzymes or kinases involved in these posttranslational
modifications in this region of the lens. The molecular mechanisms involved in
this process, including how ZO-1 translocates from the narrow faces of fiber
cells to gap junctions, remain to be elucidated.
The results of FL experiments provided direct evidence that ZO-1 is
distributed in close topographic association with 3Cx46 and
8Cx50 connexins. Furthermore, these experiments show that the
interaction between ZO-1 and connexins is resistant to mild SDS treatment.
Junctional constituents probably form a stable scaffold associated
specifically with sites of initiation and progressive packing of the
junctional domains. However, we cannot exclude the presence of an SDS-soluble
pool of ZO-1 that is not revealed by FL and that could be associated with
other membrane domains. This could explain the apparent discrepancy between
our FL and immunofluorescence results concerning the overall localization of
ZO-1, because the latter technique, using chemically fixed sections, showed
additional labeling of ZO-1 at nonjunctional membrane domains of fiber
cells.
During elongation and terminal differentiation of the lens fibers, several
membrane and cytoskeletal proteins are expressed, in particular, the major
transmembrane protein of the fibers, MP26 (MIP or Aquaporin 0), which has a
dual function of water transporter and adhesion molecule
(Benedetti et al.,
2000). FL experiments have demonstrated that MP26, during the
packing of 3Cx46 and 8Cx50 connexons, forms a belt of
transmembrane-linked pairs around the junctional plaques. FL of ZO-1 shows
that this protein does not form a crown around the junctional plaque but
rather appears scattered randomly within the plaque surface. This topographic
distribution is suggestive of a more general role in gap junction assembly,
such as interacting with cytoskeletal constituents and/or recruiting of
signaling molecules to the junctional domain.
FL experiments on lens fiber cells of 3Cx46 or 8Cx50 connexin
knock-out mice showed that either connexin can be associated with ZO-1. Hence,
in agreement with our biochemical data, the presence of heteromeric and/or
heterotypic connexons is not required for the ZO-1–connexin
interaction.
Molecular Interactions between ZO-1 and 3Cx46 and 8Cx50
Connexins
The molecular mechanism of ZO-1 interaction with 3Cx46 and
8Cx50 is apparently similar to the interaction described for
1Cx43, involving the second PDZ domain of ZO-1 and the most
COOH-terminal connexin residues (Giepmans
and Moolenaar, 1998; Giepmans
et al., 2001). The consensus motifs for PDZ-binding are
the COOH-terminal sequences E-S/T-X-V/I (type I), 
-X- (type II),
/
-X- (type III) (Songyang
et al., 1997; Dev
et al., 2001), and X-D-X-V (type IV
[Sheng and Sala, 2001]), where
is a hydrophobic residue and is a basic residue. 1Cx43 and
mouse 3Cx46 and 8Cx50 connexins have a potential type II
PDZ-binding domain at their COOH-termini. Furthermore, the COOH-terminal
sequences of these connexins have similar but not identical residues conserved
between species. For example, the COOH-terminal sequence of 3Cx46
connexin is D-L-A-I in human and rat, whereas it is
D-L-A-V in Cx56, the chicken orthologue of 3Cx46. The same
V-I exchange can be found in orthologues of 8Cx50, where the human
sequence is D-L-T-V, whereas the mouse sequence is
D-L-T-I. Because both valine and isoleucine residues in position 0
can bind type I PDZ-domains, we would not expect these sequence variations to
affect binding to ZO-1.
We have recently shown that 11/Cx31.9 connexin binds to ZO-1 via a
similar mechanism (Nielsen et
al., 2002), and this is likely to be the case for
7Cx45 connexin as well (Kausalya
et al., 2001; Laing
et al., 2001). The human connexin family contains 20
members (Willecke et al.,
2002), and alignment study of the COOH-termini revealed that 9
members, none of which are from the -class, contain potential ZO-1
binding motifs (Nielsen et al.,
2001). Because the molecular mechanism of ZO-1 binding appears
similar in several connexins, there is a possibility that the biological
function(s) of this interaction could also be similar for the different
connexin isotypes.
Interactions of ZO-1 with Truncated Forms of 3Cx46 and
8Cx50 Connexins
Our results indicate that the truncated forms of 3Cx46 and
8Cx50 lacking the ZO-1 binding domains can still traffic and form
structures between adjacent cells when expressed in nonpolarized HEK293 cells.
This is in accordance with previous reports describing the expression of
truncated forms of connexins.
A truncated form of 8Cx50 lacking the COOH-terminal domain forms gap
junction channels with properties similar to wild-type channels, except for
loss of pHi sensitivity (Xu
et al., 2002). Truncated forms of 1Cx43 are also
known to be able to form functional gap junctions when overexpressed in
nonpolarized cells lines (Fishman et
al., 1991; Unger et
al., 1999). In contrast, 7Cx45 mutants lacking the
ZO-1 binding site were reported not to localize to sites of cell–cell
contact in polarized MDCK cells (Kausalya
et al., 2001). This discrepancy may be related to
differences between polarized and nonpolarized cells. Another possibility is
that the functional role of the ZO-1 interaction is dependent on the connexin
isotype.
ZO-1 Involvement in Recycling of Connexins
For 1Cx43, mutants that no longer bind ZO-1 have been shown to
exhibit an increased turnover rate
(Toyofuku et al.,
2001). Furthermore, it has recently been shown that myocyte
dissociation, which is known to promote gap junction remodeling, increased the
association of ZO-1 with 1Cx43
(Barker et al., 2002).
These results suggest that the interaction of ZO-1 with 1Cx43 is
involved in regulating the recycling of the connexin. A similar biological
function has been ascribed to the interaction of the PDZ domain containing
protein EBP50 (ezrinradixin–moesin–binding phosphoprotein-50) with
2-adrenergic receptors. Disruption of this interaction
inhibits 2-adrenergic receptor recycling at the plasma
membrane and leads to missorting of endocytosed 2-adrenergic
receptors to lysosomes (Hall et
al., 1998b). The lens is characterized by a slow but constant
connexin turnover rate detected primarily at the equatorial cortical region,
where the fiber junctions are assembled. ZO-1 may have a similar role in
recycling of fiber gap junctions in this region of the lens.
The lens may serve as an excellent model system for studying these interactions because of its inherent properties. These include the possibilities of studying an intact, nonvital organ that expresses only a limited number of well-characterized connexins and the availability of knockout mice lacking combinations of all connexins expressed in the lens.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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# Present addresses: Bascom Palmer Eye Institute, University of Miami School
of Medicine, Miami, FL 33136
** Corresponding author. E-mail address: nalin{at}uic.edu.
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REFERENCES |
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